High-throughput and highly multiplexed imaging with programmable nucleic acid probes

ABSTRACT

The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets of interest.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/108,911, filed Jun. 29, 2016, which is a national stage filing under35 U.S.C. § 371 of international application number PCT/US2015/020034,filed Mar. 11, 2015, which was published under PCT Article 21(2) inEnglish and claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 61/951,461, filed Mar. 11, 2014, theentire contents of each of which are incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with government support under 1DP2OD007292-01,5R01EB018659-02, and 1-U01-MH106011-01 awarded by National Institutes ofHealth; and under N00014-13-1-0593 awarded by U.S. Department of DefenseOffice of Naval Research; and under CCF-1317291 awarded by NationalScience Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the field of detection andquantification of analytes (e.g., targets).

BACKGROUND OF THE INVENTION

Fluorescence microscopy is a powerful tool for exploring molecules in,for example, a biological system. However, the number of distinctspecies that can be distinguishably and simultaneously visualized (i.e.the multiplexing power) is limited by the spectral overlap between thefluorophores.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, methods and compositions fordetecting, imaging and/or quantitating targets (e.g., biomolecules) ofinterest. Some of the methods provided herein involve (1) contacting asample to be analyzed (e.g., a sample suspected of containing one ormore targets of interest) with moieties that bind specifically to thetargets (each moiety being a binding partner of a given target), whereineach moiety is conjugated to a nucleic acid (referred to herein as adocking strand) and wherein binding partners of different specificityare conjugated to different docking strands, (2) optionally removingunbound binding partners, (3) contacting the sample with labeled (e.g.,fluorescently labeled) nucleic acids having a nucleotide sequence thatis complementary to and thus specific for one docking strand (suchlabeled nucleic acids referred to herein as labeled imager strands), (4)optionally removing unbound imager strands, (5) imaging the sample inwhole or in part to detect the location and number of bound imagerstrands, (6) extinguishing signal from the labeled imager strand fromthe sample (e.g., by bleaching, including photobleaching), and (7)repeating steps (3)-(6) each time with an imager strand having a uniquenucleotide sequence relative to all other imager strands used in themethod.

Imager strands may be identically labeled, including identicallyfluorescently labeled. In other embodiments, imager strands having anidentical sequence may be identically labeled. The first approach may bemore convenient as it requires a single excitation wavelength anddetector.

In this manner, it is possible to detect, image and/or quantitate two ormore targets in a sample, regardless of their location in the sample,including regardless of whether their location in the sample is so closetogether to be indistinguishable if signal from the two or more targetswas observed simultaneously. Thus, the distance between two or moretargets may be below the resolution distance of the imaging system usedto detect the targets, and still using the methods provided herein itwould be possible to distinguish the two or more targets from eachother, thereby facilitating a more accurate and robust detection andquantitation of such targets. In some instances, the resolution distancemay be about 50 nm, as an example.

It is to be understood that the “target content” of a sample may beknown or suspected, or unknown and unsuspected, prior to performing themethod. The binding partners contacting the sample may bind to thesample, or they may not, depending on whether the target is present orabsent (e.g., when the target is present, the binding partner may bindto the sample). The imager strands contacting the sample may bind to thesample, or they may not, depending on whether the target is present orabsent (e.g., when the target is present, the imager strand may bind acorresponding docking strand bound to the target). “Binding to thesample” means that the binding partner or the imager strand is bound toits respective target or docking strand.

The binding partners may be protein in nature, such as antibodies orantibody fragments. In the context of a binding partner that is anantibody or antibody fragment, the docking strands may be conjugatedthereto at a constant region. The binding partner may be an antibodysuch as a monoclonal antibody, or it may be an antigen-binding antibodyfragment such as an antigen-binding fragment from a monoclonal antibody.In some embodiments, the binding partner is a receptor.

The binding partner may be linked to the docking strand through anintermediate linker. In some embodiments, an intermediate linkercomprises biotin and/or streptavidin.

The imager strands may be fluorescently labeled (i.e., they areconjugated to a fluorophore). Fluorophores conjugated to imager strandsof different nucleotide sequence may be identical to each other, or theymay have an emission profile that overlaps or that doesn't overlap withthat of other fluorophores. The fluorescently labeled imager strand maycomprise at least one fluorophore.

In some instances, fluorescently labeled imager nucleic acids such asimager strands may comprise 1, 2, 3, or more fluorophores.

The sample may be a cell, a population of cells, or a cell lysate from acell or a population of cells. The target may be a protein.

It will therefore be appreciated that the invention provides a methodfor detecting analytes by binding analytes to their respective bindingpartners and sequentially determining the presence of such bindingpartners, by repeatedly binding, detecting and extinguishing (e.g.,bleaching, such as photobleaching) imager strands, that optionally areidentically labeled (e.g., identically fluorescently labeled).

Accordingly, the disclosure provides a method comprising (1) contactinga sample being tested for the presence of one or more targets with oneor more target-specific binding partners, wherein each target-specificbinding partner is linked to a docking strand, and whereintarget-specific binding partners of different specificity are linked todifferent docking strands, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager strands having a nucleotide sequence that is complementary to adocking strand, (4) optionally removing unbound labeled imager strands,(5) imaging the sample to detect location and number of bound labeledimager strands, (6) extinguishing signal from the bound labeled imagerstrand, and (7) repeating steps (3)-(6), each time with a labeled imagerstrand having a unique nucleotide sequence relative to all other labeledimager strands.

In some embodiments, the sample is contacted with more than onetarget-specific binding partner in step (1).

In some embodiments, the target-specific binding partner is an antibodyor an antibody fragment.

In some embodiments, the labeled imager strands are labeled identically.In some embodiments, the labeled imager strands each comprise a distinctlabel. In some embodiments, the labeled imager strands are fluorescentlylabeled imager strands.

In some embodiments, the one or more targets are proteins. In someembodiments, the sample is a cell, a cell lysate or a tissue lysate.

In some embodiments, the sample is imaged in step (5) using confocal orepi-fluorescence microscopy.

In some embodiments, extinguishing signal in step (6) comprisesphotobleaching.

The disclosure further provides a composition comprising a sample boundto more than one target-recognition moieties such as target-specificbinding partners, each target-recognition moiety bound to a dockingnucleic acid such as a docking strand, and at least one docking nucleicacid stably bound to a labeled imager nucleic acid such as an imagerstrand.

The disclosure further provides a composition comprising a sample boundto more than one target-specific binding partners, each binding partnerbound to a docking strand, and at least one docking strand stably boundto a labeled imager strand.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-recognition moieties such as target-specific bindingpartners, wherein each target-recognition moiety is linked to a dockingnucleic acid such as a docking strand, and wherein target-recognitionmoieties of different specificity are linked to different dockingnucleic acids, (2) optionally removing unbound target-recognitionmoieties, (3) contacting the sample with labeled imager nucleic acidssuch as imager strands having a nucleotide sequence that iscomplementary to a docking nucleic acid, (4) optionally removing unboundlabeled imager nucleic acids, (5) imaging the sample to detect locationand number of bound labeled imager nucleic acids, (6) removing the boundlabeled imager nucleic acids from the docking nucleic acids by alteringtemperature and/or buffer condition, and (7) repeating steps (3)-(6),each time with a labeled imager nucleic acid having a unique nucleotidesequence relative to all other labeled imager nucleic acids. The imagernucleic acid dissociates from the docking nucleic acid spontaneouslyunder such conditions.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-specific binding partners, wherein each target-specificbinding partner is linked to a docking nucleic acid, and whereintarget-specific binding partners of different specificity are linked todifferent docking nucleic acids, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager nucleic acids having a nucleotide sequence that is complementaryto a docking nucleic acid, (4) optionally removing unbound labeledimager nucleic acids, (5) imaging the sample to detect location andnumber of bound labeled imager nucleic acids, (6) removing the boundlabeled imager nucleic acids from the docking nucleic acids by alteringtemperature and/or buffer condition, and (7) repeating steps (3)-(6),each time with a labeled imager nucleic acid having a unique nucleotidesequence relative to all other labeled imager nucleic acids. The imagernucleic acid dissociates from the docking nucleic acid spontaneouslyunder such conditions.

In some embodiments, the labeled imager nucleic acids are removed fromthe docking nucleic acids by decreasing salt concentration, addition ofa denaturant, or increasing temperature. In some embodiments, the saltis Mg++. In some embodiments, the denaturant is formamide, urea or DMSO.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-recognition moieties such as target-specific bindingpartners, wherein each target-recognition moiety is linked to a dockingnucleic acid such as a docking strand, and wherein target-recognitionmoieties of different specificity are linked to different dockingnucleic acids, (2) optionally removing unbound target-recognitionmoieties, (3) contacting the sample with labeled imager nucleic acidssuch as imager strands having a nucleotide sequence that iscomplementary to a docking nucleic acid, (4) optionally removing unboundlabeled imager nucleic acids, (5) imaging the sample to detect locationand number of bound labeled imager nucleic acids, (6) removing the boundlabeled imager nucleic acids from the docking nucleic acids, and (7)repeating steps (3)-(6), each time with a labeled imager nucleic acidhaving a unique nucleotide sequence relative to all other labeled imagernucleic acids.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-specific binding partners, wherein each target-specificbinding partner is linked to a docking nucleic acid, and whereintarget-specific binding partners of different specificity are linked todifferent docking nucleic acids, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager nucleic acids having a nucleotide sequence that is complementaryto a docking nucleic acid, (4) optionally removing unbound labeledimager nucleic acids, (5) imaging the sample to detect location andnumber of bound labeled imager nucleic acids, (6) removing the boundlabeled imager nucleic acids from the docking nucleic acids, and (7)repeating steps (3)-(6), each time with a labeled imager nucleic acidhaving a unique nucleotide sequence relative to all other labeled imagernucleic acids.

In some embodiments, in step (6) the labeled imager nucleic acids arenot removed from the docking nucleic acids by strand displacement in thepresence of a competing nucleic acid.

In some embodiments, in step (6) the labeled imager nucleic acids areremoved from the docking nucleic acids by chemically, photochemically,or enzymatically cleaving, modifying or degrading the labeled imagernucleic acids.

In some embodiments, when the labeled imager nucleic acid is bound toits respective docking nucleic acid, there is no single-stranded regionon the imager nucleic acid or the docking nucleic acid. In someembodiments, the docking nucleic acid does not have a toehold sequence.In some embodiments, the imager nucleic acid does not have a toeholdsequence.

In some embodiments, the labeled imager nucleic acid is notself-quenching.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-recognition moieties such as target-specific bindingpartners, wherein each target-recognition moiety is linked to a dockingnucleic acid such as a docking strand, and wherein target-recognitionmoieties of different specificity are linked to different dockingnucleic acids, (2) optionally removing unbound target-recognitionmoieties, (3) contacting the sample with labeled imager nucleic acidssuch as imager strands having a nucleotide sequence that iscomplementary to a docking nucleic acid, (4) optionally removing unboundlabeled imager nucleic acids, (5) imaging the sample to detect locationand number of bound labeled imager nucleic acids, (6) inactivating thebound labeled imager nucleic acids, by removing or modifying theirsignal-emitting moieties without removing the imager nucleic acid in itsentirety, and (7) repeating steps (3)-(6), each time with a labeledimager nucleic acids having a unique nucleotide sequence relative to allother labeled imager nucleic acids.

The disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-specific binding partners, wherein each target-specificbinding partner is linked to a docking nucleic acid, and whereintarget-specific binding partners of different specificity are linked todifferent docking nucleic acids, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager nucleic acids having a nucleotide sequence that is complementaryto a docking nucleic acid, (4) optionally removing unbound labeledimager nucleic acids, (5) imaging the sample to detect location andnumber of bound labeled imager nucleic acids, (6) inactivating the boundlabeled imager nucleic acids, by removing or modifying theirsignal-emitting moieties without removing the imager nucleic acid in itsentirety, and (7) repeating steps (3)-(6), each time with a labeledimager nucleic acids having a unique nucleotide sequence relative to allother labeled imager nucleic acids.

Various embodiments apply equally to the afore-mentioned methods. Theseembodiments are as follows:

In some embodiments, the sample is contacted with more than onetarget-specific binding partner in step (1). In some embodiments, thetarget-specific binding partner is an antibody or an antibody fragment.In some embodiments, the target-specific binding partner is a natural orengineered ligand, a small molecule, an aptamer, a peptide or anoligonucleotide.

In some embodiments, the labeled imager nucleic acids are labeledidentically. In some embodiments, the labeled imager nucleic acids eachcomprise a distinct label. In some embodiments, the labeled imagernucleic acids are fluorescently labeled imager nucleic acids.

In some embodiments, the one or more targets are proteins. In someembodiments, the sample is a cell, a cell lysate or a tissue lysate.

In some embodiments, the sample is imaged in step (5) using confocal orepi-fluorescence microscopy.

In some embodiments, the unbound docking nucleic acid is partiallydouble-stranded. In some embodiments, the unbound imager nucleic acid ispartially double-stranded.

In some embodiments, the imager nucleic acid is a molecular beacon orcomprises a hairpin secondary structure. In some embodiments, the imagernucleic acid is a molecular beacon or comprises a hairpin secondarystructure that is self-quenching. In some embodiments, the imagernucleic acid is a hemiduplex. In some embodiments, the hemiduplex isself-quenching. In some embodiments, the imager nucleic acid is bound tomultiple signal-emitting moieties through a dendrimeric structure or apolymeric structure. The imager nucleic acid may be linear or branched.

In some embodiments, the docking nucleic acid comprises a hairpinsecondary structure.

These and other embodiments will be described in greater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of one embodiment of a high-throughput andintrinsically scalable multiplexed imaging approach provided in thisdisclosure. Cells are imaged after probe hybridization and thenphotobleached before a subsequent round of imaging.

FIG. 2 is a schematic of one embodiment of a high-throughput andintrinsically scalable multiplexed imaging approach based on bufferexchange using solutions with slight denaturation characteristics, suchas decreased salt concentration, increased formamide concentration, orhigher temperature.

FIG. 3 is a schematic of one embodiment of inactivation of the imagerstrand by removing the imager strand using the methods provided in thisdisclosure.

FIG. 4 is a schematic of one embodiment of inactivation of the imagerstrand by inactivating the fluorophore without removing the nucleic acidportion of the imager strand.

FIG. 5 is a schematic of one embodiment of a molecular beacon-likeself-quenching imager strand.

FIG. 6 is a schematic of one embodiment of a hemi-duplex self-quenchingimager strand.

FIG. 7 is a schematic of one embodiment of a non-single-stranded dockingstrand.

FIG. 8 is a schematic of one embodiment of an imager strand thatrecruits multiple copies of the signal-emitting moieties to the dockingstrand.

FIG. 9 is a schematic of one embodiment of a non-single-stranded imagerstrand.

FIG. 10 is a graph showing the predicted dissociation constants of 10different oligonucleotides with the respective reverse-complementarystrands at 3 different conditions.

Sequence a1: (SEQ ID NO: 1) 5′-CATCTAAAGCC-3′; Sequence a2:(SEQ ID NO: 2) 5′-GAATTTCCTCG-3′; Sequence a3: (SEQ ID NO: 3)5′-GTTTAATTGCG-3′; Sequence a4: (SEQ ID NO: 4) 5′-ACAATTCTTCG-3′;Sequence a5: (SEQ ID NO: 5) 5′-TTTCTTGCTTC-3′; Sequence a6:(SEQ ID NO: 6) 5′-GCATTGTTACT-3′; Sequence a7: (SEQ ID NO: 7)5′-ATATACAAGCG-3′; Sequence a8: (SEQ ID NO: 8) 5′-GCTGTCTATTG-3′;Sequence a9: (SEQ ID NO: 9) 5′-TCTTTATGCTG-3′; Sequence a10:(SEQ ID NO: 10) 5′-CAATCTCATCC-3′.

DESCRIPTION OF THE INVENTION

The invention provides, inter alia, compositions and methods formultiplexed fluorescence imaging, for example, in a cellular environmentusing nucleic acid-based imaging probes (e.g., DNA-based imagingprobes). Methods provided herein are based, in part, on theprogrammability of nucleic acid docking strands and imager strands. Thatis, for example, docking strands and imager strands can be designed suchthat they bind to each other under certain conditions for a certainperiod of time. This programmability permits stable binding of imagerstrands to docking strands, as provided herein. Generally, the methodsprovided herein are directed to identifying one or more target(s) (e.g.,biomolecule(s) such as a protein or nucleic acid) in a particular sample(e.g., biological sample). In some instances, whether or not one or moretarget(s) is present in sample is unknown. Thus, methods of the presentdisclosure may be used to determine the presence or absence of one ormore target(s) in a sample suspected of containing the target(s). In anyone of the aspects and embodiments provided herein, a sample may containor may be suspected of containing one or more target(s).

Thus, the invention provides methods for performing high-throughput andhighly multiplexed imaging and analyte/target detection based onprogrammable nucleic acid (e.g., DNA) probes. These methods rely on asequential imaging approach employing orthogonal imager strands that canstably attach to a complementary docking strand immobilized on bindingpartners, such as antibodies (FIG. 1). After hybridization and imagingwith an imager strand, an extinguishing step (such as a photobleachingstep) is performed to eliminate and/or reduce fluorescence from thehybridized (bound) imager strands.

In another embodiment, the methods utilize weaker binding betweendocking and imaging strands in order to remove signal. For example, thehybridization conditions may be changed such that the melting point ofthe duplex that is formed between the docking or imager strands isslightly above room temperature (e.g., 25° C.) or the imagingtemperature. The labeling step (i.e., the step at which the imagerstrands are bound to their respective docking strands) and the imagingstep are performed as described above. As an example, after the firsttarget is imaged, the sample is subjected to a denaturing condition. Thedenaturing condition may be provided in a buffer exchange step using asolution with for example lower salt concentration, presence of orincrease in the concentration of a denaturant such as formamide, orincreased temperature (FIG. 2). The sample may be alternatively oradditionally exposed to an increased temperature. The aforementionedincreases or decreases are relative to the conditions existing at thelabeling step (i.e., when the imager strand is bound to the dockingstrand). In the case of the buffer exchange, the sample may be washed,the buffer exchange may be repeated, the sample may be washed again, andthen the next imager strand may be added to the sample.

For multiplexing, different reservoirs of orthogonal imager strands aresequentially applied after every step of, for example, photobleaching orother method for extinguishing signal or imager strand inactivation orremoval to the same sample in order to potentially image an infinitenumber of targets. Unlike traditional imaging approaches, wheremultiplexing is limited by spectral overlap between color channels, themethods provided herein are only limited by the number of possibleorthogonal nucleotide sequences (of the docking strands or alternativelythe imager strands). As a larger number of orthogonal nucleotidesequences can be readily designed, this approach has intrinsicallyscalable multiplexing capability just by using a single fluorophore.This method can be readily integrated with standard microscopy setups(e.g., confocal or epi-fluorescence microscopes), allowing highthroughput analysis of the sample.

The methods have applicability in, for example, high-throughputscreening assays such as drug screening assays. This imaging approachallows analysis of large populations of cells (˜1,000-10,000) or tissuesamples in an ultra-multiplexed format while imaging using standardconfocal or epi-fluorescence microscope. Screening large numbers oftargets such as proteins from the same sample in a high-throughputmanner will provide information about new drugs or modifiers whileproviding cellular heterogeneity information. The large scale screeningof tissue samples with high-throughput and ultra-multiplexed imagingcapabilities will be useful in pathology analysis, for example, in ahospital or other service provider setting.

Methods provided herein can also be used to identify the absolutequantity of a single target (e.g., such as, for example, a particularprotein), or the quantity of a single target relative to one or moreother targets.

Further, methods provided herein may be used to identify the location ofa target within a sample or relative to other targets in the sample.

This disclosure therefore provides a method comprising (1) contacting asample simultaneously with a plurality of sequence-labeledtarget-recognition moieties, (2) introducing imager nucleic acids suchas imager strands recognizing, through sequence complementarity, asubset of docking nucleic acids such as docking strands in thesequence-labeled target-recognition moieties, (3) removing orinactivating the imager nucleic acids or extinguishing signal from theimager nucleic acids, and (4) repeating step (2) and optionally step (3)at least once in order to image and detect one or more additionaldocking nucleic acids.

The method may optionally comprise labeling a plurality oftarget-recognition moieties with docking nucleic acids such as dockingstrands to form sequence-labeled target-recognition moieties.

This disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-specific binding partners, wherein each target-specificbinding partner is linked to a docking strand, and whereintarget-specific binding partners of different specificity are linked todifferent docking strands, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager strands having a nucleotide sequence that is complementary to adocking strand, (4) optionally removing unbound labeled imager strands,(5) imaging the sample to detect location and number of bound labeledimager strands, (6) extinguishing signal from the bound labeled imagerstrand, and (7) repeating steps (3)-(6), each time with a labeled imagerstrand having a unique nucleotide sequence relative to all other labeledimager strands.

Steps (3)-(6) may be repeated once or multiple times. For example, steps(3)-(6) may be repeated 1-10 times or more. In some embodiments, steps(3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

This disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-recognition moieties such as target-specific bindingpartners, wherein each target-recognition moiety is linked to a dockingnucleic acid, and wherein target-recognition moieties of differentspecificity are linked to different docking nucleic acids, (2)optionally removing unbound target-recognition moieties, (3) contactingthe sample with labeled imager nucleic acids such as imager strandshaving a nucleotide sequence that is complementary to a docking nucleicacid, (4) optionally removing unbound labeled imager nucleic acids, (5)imaging the sample to detect location and number of bound labeled imagernucleic acids, (6) removing the bound labeled imager nucleic acids fromthe docking nucleic acids, and (7) repeating steps (3)-(6), each timewith a labeled imager nucleic acid having a unique nucleotide sequencerelative to all other labeled imager nucleic acids.

Steps (3)-(6) may be repeated once or multiple times. For example, steps(3)-(6) may be repeated 1-10 times or more. In some embodiments, steps(3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

This disclosure further provides a method comprising (1) contacting asample being tested for the presence of one or more targets with one ormore target-recognition moieties such as target-specific bindingpartners, wherein each target-recognition moieties is linked to adocking nucleic acid such as a docking strand, and whereintarget-recognition moieties of different specificity are linked todifferent docking nucleic acids, (2) optionally removing unboundtarget-recognition moieties, (3) contacting the sample with labeledimager nucleic acids such as imager strands having a nucleotide sequencethat is complementary to a docking nucleic acid, (4) optionally removingunbound labeled imager nucleic acids, (5) imaging the sample to detectlocation and number of bound labeled imager nucleic acids, (6)inactivating the bound labeled imager nucleic acids, by removing ormodifying their signal-emitting moieties without removing the imagernucleic acid in its entirety, and (7) repeating steps (3)-(6), each timewith a labeled imager nucleic acid having a unique nucleotide sequencerelative to all other labeled imager nucleic acids.

Steps (3)-(6) may be repeated once or multiple times. For example, steps(3)-(6) may be repeated 1-10 times or more. In some embodiments, steps(3)-(6) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

In some embodiments, the methods provided herein include a step ofremoving an imager nucleic acid such as an imager strand that is boundto a docking nucleic acids such as a docking strand, using a methodother than strand displacement.

In some embodiments, the methods provided herein include a step ofremoving an imager nucleic acid such as an imager strand that is boundto a docking nucleic acid such as a docking strand, wherein the imagernucleic acid emits signal (i.e., such signal is not quenched) prior tobinding to the docking nucleic acid.

In some embodiments, the methods provided herein include a step ofremoving an imager nucleic acid such as an imager strand that is boundto a docking nucleic acid such as a docking strand, wherein the imagernucleic acid is removed using a nucleic acid that does not comprise aquencher.

In each of the foregoing methods, the docking nucleic acid including thedocking strand may be a single-stranded docking nucleic acid or dockingstrand, or it may be a double-stranded docking nucleic acid or dockingstrand, or it may be a partially double-stranded docking nucleic acid ordocking strand (e.g., containing a single-stranded and a double-strandedregion).

In some embodiments, where a plurality of target-recognition moieties,including a plurality of binding partners, are used, the plurality maybe contacted with the sample, and thus with targets of interest,simultaneously. The target-recognition moieties such as the bindingpartners need not be contacted with the sample sequentially, althoughthey can be.

These various methods facilitate high throughput imaging with spinningdisk confocal microscopy. It is estimated that a one color whole cell 3Dimaging process would take on average about 30 seconds. The methodallows for imaging of large areas (e.g., up to mm scale) with compatible10× or 20× objective. An imaging depth of about 30-50 microns may beachieved. The methods provided herein have been used to stain actin,Ki-67, clathrin, cytokeratin, among others (data not shown).

Binding Partners

The methods employ binding partners conjugated to nucleic acids (e.g.,docking nucleic acids such as docking strands). These may be referred toherein as binding partner-nucleic acid conjugates (“BP-NA conjugates”).They may also be referred to as sequence-labeled target-recognitionmoieties. As used herein, “binding partner-nucleic acid conjugate,” or“BP-NA conjugate,” refers to a molecule linked (e.g., through anN-Hydroxysuccinimide (NHS) linker) to a single-stranded nucleic acid(e.g., DNA) docking strand.

The binding partner of the conjugate may be any moiety (e.g., antibodyor aptamer) that has an affinity for (e.g., binds to) a target, such asa biomolecule (e.g., protein or nucleic acid), of interest. In someembodiments, the binding partner is a protein. BP-NA-conjugates thatcomprise a protein (or peptide) linked to a docking strand may bereferred to herein as “protein-nucleic acid conjugates,” or “protein-NAconjugates.” Examples of proteins for use in the conjugates of theinvention include, without limitation, antibodies (e.g., monoclonalantibodies), antigen-binding antibody fragments (e.g., Fab fragments),receptors, peptides and peptide aptamers. Other binding partners may beused in accordance with the invention. For example, binding partnersthat bind to targets through electrostatic (e.g., electrostaticparticles), hydrophobic or magnetic (e.g., magnetic particles)interactions are contemplated herein.

As used herein, “antibody” includes full-length antibodies and anyantigen binding fragment (e.g., “antigen-binding portion”) or singlechain thereof. The term “antibody” includes, without limitation, aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Antibodies may be polyclonal or monoclonal; xenogeneic,allogeneic, or syngeneic; or modified forms thereof (e.g., humanized,chimeric).

As used herein, “antigen-binding portion” of an antibody, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen. The antigen-binding function of an antibody can beperformed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(H), V_(L), C_(L) and C_(H1) domains; (ii) a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(H) and V_(L)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341:544 546, 1989), which consists of a V_(H) domain; and (vi) anisolated complementarity determining region (CDR) or (vii) a combinationof two or more isolated CDRs, which may optionally be joined by asynthetic linker. Furthermore, although the two domains of the Fvfragment, V_(H) and V_(L), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(H) and V_(L)regions pair to form monovalent molecules (known as single chain Fv(scFv); see, e.g., Bird et al. Science 242:423 426, 1988; and Huston etal. Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single chainantibodies are also encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

As used herein, “receptors” refer to cellular-derived molecules (e.g.,proteins) that bind to ligands such as, for example, peptides or smallmolecules (e.g., low molecular weight (<900 Daltons) organic orinorganic compounds).

As used herein, “peptide aptamer” refers to a molecule with a variablepeptide sequence inserted into a constant scaffold protein (see, e.g.,Baines I C, et al. Drug Discov. Today 11:334-341, 2006).

In some embodiments, the molecule of the BP-NA conjugate is a nucleicacid such as, for example, a nucleic acid aptamer. As used herein,“nucleic acid aptamer” refers to a small RNA or DNA molecules that canform secondary and tertiary structures capable of specifically bindingproteins or other cellular targets (see, e.g., Ni X, et al. Curr MedChem. 18(27): 4206-4214, 2011). Thus, in some embodiments, the BP-NAconjugate may be an aptamer-nucleic acid conjugate.

Some embodiments of the invention use target-recognition moieties toidentify and label targets. Target-recognition moieties are agents thatspecifically recognize targets of interest in the sample. Examples oftarget-recognition moieties include binding partners such as thoserecited herein. Target-recognition moieties include antibodies, antibodyfragments and antibody derivatives such as single-chain antibodies,single-chain Fv domains, Fab domains, nanobodies, and the like,peptides, aptamers, and oligonucleotides (e.g., to detect nucleic acidsof interest in procedures such as fluorescence in situ hybridization, orFISH).

Docking Nucleic Acids such as Docking Strands

Certain embodiments of the invention may refer to docking nucleic acids.Docking nucleic acids include docking strands as described herein.Docking nucleic acids are linear nucleic acids capable of binding to anucleic acid having a complementary sequence (such as an imager nucleicacid). A docking nucleic acid may be comprised of or may consist of DNA,RNA, or nucleic acid-like structures with other phosphate-sugarbackbones (e.g. 2′-O-methyl RNA, 2′-fluoral RNA, LNA, XNA) or backbonescomprising non-phosphate-sugar moieties (e.g., peptide nucleic acid andmorpholino). The nucleobases may include naturally occurring nucleobasessuch as adenine, thymine, guanine, cytosine, inosine, and theirderivatives, as well as non-naturally occurring nucleobases such asisoC, isoG, dP and dZ. A docking nucleic acid, when not bound to itscomplementary imager nucleic acid, may be single-stranded without stablesecondary structure. Alternatively, the docking nucleic acid maycomprise secondary structure such as a hairpin loop (FIG. 7, top). Adocking nucleic acid may be part of a multi-strand complex (FIG. 7,bottom).

As used herein, a “docking strand” refers to a single-stranded nucleicacid (e.g., DNA) capable of stably binding to its complementary imagerstrands. Stable binding may be a result of the length of the dockingstrand (and conversely the imager strand) or it may be the result of theparticular conditions under which hybridization occurs (e.g., saltconcentration, temperature, etc.). In some embodiments, a docking strandis about 20 to about 60, or more, nucleotides in length. A dockingstrand may be capable of binding to one or more identical imager strands(of identical sequence and identically labeled).

Imager Nucleic Acids such as Imager Strands

Certain embodiments of the invention may refer to imager nucleic acids.Imager nucleic acids include imager strands as described herein. Imagernucleic acids are nucleic acids that can (1) interact with a dockingnucleic acid via sequence-specific complementarity and (2) recruit asignal-emitting moiety or multiple copies of signal-emitting moieties bycovalent or non-covalent interactions. The imager nucleic acids may belinear or branched as described herein. One imager nucleic acid mayrecruit multiple copies of the signal-emitting moiety via a polymeric(FIG. 8, top) or dendrimeric structure (FIG. 8, bottom). For example, apolymeric or dendrimeric structure can be synthesized chemically usingmethods such as those discussed in Nazemi A. et al. Chemistry ofBioconjugates: Synthesis, Characterization, and Biomedical Applications,Published Online: 13 Feb. 2014) and references provided therein.Alternatively, the polymeric or dendrimeric structure can be formed byDNA hybridization as shown, for example, in Dirks R. et al. Proc. Nat.Acad. Sci. U.S.A., 2004; 1010(43):15275-78; and in Um S. H. et al. Nat.Protocols 2006; 1:995-1000, each of which is incorporated by referenceherein.

An imager nucleic acid may be comprised of or may consist of DNA, RNA,or nucleic acid-like structures with other phosphate-sugar backbones(e.g. 2′-O-methyl RNA, 2′-fluoral RNA, LNA, XNA) or backbones comprisingnon-phosphate-sugar moieties (e.g., peptide nucleic acid andmorpholino). The nucleobases may include naturally occurring nucleobasessuch as adenine, thymine, guanine, cytosine, inosine, and theirderivatives, as well as non-naturally occurring nucleobases such asisoC, isoG, dP and dZ.

In some embodiments, an imager nucleic acid is about 30 to about 60nucleotides, or more, in length, including 30, 35, 40, 45, 50, 55 or 60nucleotides in length. In some embodiments, an imager nucleic acid is 30to 40, 30 to 50, 40 to 50, 40 to 60, or 50 to 60 nucleotides in length.

An imager nucleic acid, when not bound to its complementary dockingnucleic acid, may be single-stranded without stable secondary structure.Alternatively, the imager nucleic acid may comprise secondary structuresuch as a hairpin loop (FIG. 9, top). An imager nucleic acid may be partof a multi-strand complex (FIG. 9, bottom).

In some embodiments, the imager strand can be self-quenching, intendingthat the unbound imager nucleic acid may carry a quencher moiety that isin close proximity with the signal-emitting moiety such as afluorophore. To achieve this, the imager nucleic acid can be designed toadopt either a molecular beacon-like structure (FIG. 5) or a hemiduplexstructure (FIG. 6).

This self-quenching variation can be used to reduce background and/oravoid the washing step. Additionally or alternatively, the binding andimaging buffer may contain additives routinely used in FISH, NorthernBlotting and Southern Blotting (e.g., negatively charged polymers suchas dextran sulfate and heparin) to reduce non-specific binding.

A “signal-emitting moiety,” as used herein, is a moiety that, undercertain conditions, emits detectable signal, such as photon, radiation,positron, electromagnetic wave, and magnetic-nuclear resonance.

As used herein, an “imager strand” is a single-stranded nucleic acid(e.g., DNA) that is about 30 to about 60 nucleotides, or more, inlength. An imager strand of the invention is complementary to a dockingstrand and stably binds to the docking strand. Stable binding intendsthat the imager and docking strands remained bound to each other for thelength of the assay, or for at least 30 minutes, or for at least for 60minutes, or for at least for 2 hours, or more. Such binding may or maynot be reversible or irreversible.

In some embodiments, a docking nucleic acid is considered stably boundto an imager nucleic acid such as an imager strand if the nucleic acidsremain bound to each other for (or for at least) 30, 35, 40, 45, 50, 55or 60 minutes (min). In some embodiments, a docking nucleic acid isconsidered stably bound to an imager nucleic acid if the nucleic acidsremain bound to each other for (or for at least) 30 to 60 min, 30 to 120min, 40 to 60 min, 40 to 120 min, or 60 to 120 min. Such binding may ormay not be reversible, or may or may not be irreversible.

As used herein, “binding” refers to an association between at least twomolecules due to, for example, electrostatic, hydrophobic, ionic and/orhydrogen-bond interactions, optionally under physiological conditions.

Two nucleic acids, or nucleic acid domains, are “complementary” to oneanother if they base-pair, or bind, with each other to form adouble-stranded nucleic acid molecule via Watson-Crick interactions.

In some embodiments, nucleic acids of the invention such as the dockingnucleic acids and the imager nucleic acids bind to each other with“perfect complementary,” which refers to 100% complementary (e.g.,5′-ATTCGC-3′ is perfectly complementary to 5′ GCGAAT-3′).

Imager strands of the invention may be labeled with a detectable label(e.g., a fluorescent label, and thus are considered “fluorescentlylabeled”). For example, in some embodiments, an imager strand maycomprise at least one (i.e., one or more) fluorophore. Examples offluorophores for use in accordance with the invention include, withoutlimitation, xanthene derivatives (e.g., fluorescein, rhodamine, Oregongreen, eosin and Texas red), cyanine derivatives (e.g., cyanine,indocarbocyanine, oxacarbocyanine, thiacarbocyanine and merocyanine),naphthalene derivatives (e.g., dansyl and prodan derivatives), coumarinderivatives, oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole and benzoxadiazole), pyrene derivatives (e.g.,cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresylviolet and oxazine 170), acridine derivatives (e.g., proflavin, acridineorange and acridine yellow), arylmethine derivatives (e.g., auramine,crystal violet and malachite green), and tetrapyrrole derivatives (e.g.,porphin, phthalocyanine and bilirubin).

Imager nucleic acids including imager strands may be covalently labeledwith a detectable label such as those recited herein or known in theart. In some instances, imager nucleic acids including imager strandsmay comprise 2, 3, 4, or more detectable labels such as fluorophores.

Orthogonal imager nucleic acids including imager strands may comprise adistinct label (e.g., a red fluorophore, a blue fluorophore, or a greenfluorophore), or they may all comprise the same label (e.g., redfluorophores) even if they differ in nucleotide sequence.

Sequence-Labeled Target Recognition Moieties such as Binding Partner andDocking Strand Conjugates

The BP-NA conjugates (e.g., protein-nucleic acid conjugates) of theinvention may, in some embodiments, comprise an intermediate linker thatlinks (e.g., covalently or non-covalently) the binding partner to adocking strand. The intermediate linker may comprise biotin and/orstreptavidin. For example, in some embodiments, an antibody and adocking strand may each be biotinylated (i.e., linked to at least onebiotin molecule) and linked to each other through biotin binding to anintermediate streptavidin molecule. Other intermediate linkers may beused in accordance with the invention. In some embodiments, such asthose where the molecule is a nucleic acid, an intermediate linker maynot be required. For example, the docking strand of a BP-NA conjugatemay be an extension (e.g., 5′ or 3′ extension) of a nucleic acidmolecule such as, for example, a nucleic acid aptamer. Similarapproaches may be used to generate sequence-labeled target recognitionmoieties as provided herein.

Pluralities of BP-NA conjugates (e.g., protein-nucleic acid conjugates)and imager strands are provided herein. A plurality may be a populationof the same species or distinct species. A plurality of BP-NA conjugatesof the same species may comprise conjugates that all bind to the sametarget (e.g., biomolecule) (e.g., the same epitope or region/domain).Conversely, a plurality of BP-NA conjugates of distinct species maycomprise conjugates, or subsets of conjugates, each conjugate or subsetof conjugates binding to a distinct epitope on the same target or to adistinct target. A plurality of imager strands of the same species maycomprise imager strands with the same nucleotide sequence and the samefluorescent label (e.g., Cy2, Cy3 or Cy4). Conversely, a plurality ofimager strands of distinct species may comprise imager strands withdistinct nucleotide sequences (e.g., DNA sequences) and distinctfluorescent labels (e.g., Cy2, Cy3 or Cy4) or with distinct nucleotidesequences and the same fluorescent (e.g., all Cy2). The number ofdistinct species in a given plurality of BP-NA conjugates is limited bythe number of binding partners (e.g., antibodies) and the number ofdocking strands of different nucleotide sequence (and thus complementaryimager strands). In some embodiments, a plurality of BP-NA conjugates(e.g., protein-nucleic acid conjugates) comprises at least 10, 50, 100,500, 1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹ BP-NA conjugates. Likewise, in some embodiments, aplurality of fluorescently labeled imager strands comprises at least 10,50, 100, 500, 1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ fluorescently labeled imager strands. In someembodiments, a plurality may contain 1 to about 200 or more distinctspecies of BP-NA conjugates and/or imager strands. For example, aplurality may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125,150, 175, 200 or more distinct species. In some embodiments, a pluralitymay contain less than about 5 to about 200 distinct species of BP-NAconjugates and/or imager strands. For example, a plurality may containless than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 distinct species.These embodiments apply to sequence-labeled target recognition moietiesas provided herein.

Signal or Imager Nucleic Acid Inactivation

To achieve imager nucleic acid inactivation in some of the methodsprovided herein, the imager nucleic acids, including the imager strands,may be removed from the target-recognition moieties, including thebinding partners, (FIG. 3) by means such as but not limited toincreasing temperature; decreasing the concentration of counter-ions(e.g., free Mg++); introducing or increasing the concentration ofdenaturants (e.g. formamide, urea, DMSO, and the like); and chemically,photochemically or enzymatically cleaving, modifying or degrading theimager strand, or any combination thereof.

To achieve imager nucleic acid inactivation in some of the methodsprovided herein, the imager nucleic acids, including the imager strands,may be inactivated by removing and/or modifying the signal-emittingmoiety without removing the entirety of the nucleic acid portion of theimager nucleic acid from the docking strands. (FIG. 4.)

As an example, the removal of the imager nucleic acid may be facilitatedby cleaving the imager strand into multiple parts. In some embodiments,the imager nucleic acid comprises a chemically cleavable moiety that canbe cleaved by introduction of the chemical compound that acts upon suchcleavable moiety. Examples of such chemically cleavable moieties includebut are not limited to allyl groups, which can be cleaved by certainPd-based reagents (Ju J. et al., Proc Natl Acad Sci USA. 2006 Dec. 26;103(52):19635-40, incorporated by reference herein); azido groups, whichcan be cleaved by certain phosphorous-based reagents such as TCEP (Guo Jet al. Proc Natl Acad Sci USA. 2008 Jul. 8; 105(27):9145-50,incorporated by reference herein); bridging phosphorothiolates, whichcan be cleaved by silver-based reagents (Mag M. et al. Nucleic AcidsRes. 1991 Apr. 11; 19(7):1437-41, incorporated by reference herein);disulfide bonds, which can be cleaved by reducing agents such as DTT andTCEP; and ribose, which can be cleaved by a variety of nucleophiles suchas hydroxide and imidazole.

In some embodiments, the imager nucleic acid comprises a photocleavablelinker that can be cleaved photochemically (e.g., by UV exposure). Insome embodiments, the imager nucleic acid contains a moiety that can becleaved by an enzyme. Examples of such enzymatically cleavable moietiesinclude but are not limited to ribonucleotides, which can be cleaved bya variety of RNases; deoxyuridines, which can be cleaved by enzymecombinations such as USER (New England Biolabs); and restriction sites,which can be cleaved by sequence-specific nicking enzymes or restrictionenzymes. In some embodiments, the restriction enzyme may cleave both theimager nucleic acid and the docking nucleic acid. In still otherembodiments, the removal of the imager nucleic acid may be facilitatedby modifying the imager nucleic acid into a form that binds the dockingnucleic acid to form a duplex with decreased stability (or lower meltingtemperature).

As an example, the imager nucleic acid comprises azobenzene, which canbe photoisomerized, wherein different isomers affect the bindingstrength of the imager nucleic acid to the docking nucleic aciddifferently (Asanuma H. et al. Angew Chem Int Ed Engl. 2001 Jul. 16;40(14):2671-2673, incorporated by reference herein). In someembodiments, the imager nucleic acid comprises a deoxyuridine, in whichthe uracil group may be cleaved by uracil-DNA glycosylase. After theuracil is removed the binding strength of the imager strand is weakened.

Alternatively, the removal of the signal-emitting moiety can be achievedby cleaving a linker between the imager nucleic acid and thesignal-emitting moiety, if such a linker exists. Chemistries describedin herein can be used for this purpose as well.

The inactivation of the signal-emitting moiety can be achieved bychemically or photochemically modifying the signal-emitting moiety. Forexample, when the signal-emitting moiety is a fluorophore, it can bebleached by chemical agents (such as for example hydrogen peroxide,Gerdes M. et al. Proc Natl Acad Sci USA. 2013 Jul. 16; 110(29):11982-87,incorporated by reference herein) or photobleached (e.g., using softmultiwavelength excitation as described in Schubert W. et al. Nat.Biotech. 2006; 24:1270-78, incorporated by reference herein).

As will be understood in the art, “photobleaching” refers to thephotochemical alteration of a dye or a fluorophore molecule such that itis unable to fluoresce. This is caused by cleavage of covalent bonds ornon-specific reactions between the fluorophore and surroundingmolecules. Loss of activity caused by photobleaching can be controlled,in some embodiments, by reducing the intensity or time-span of lightexposure, by increasing the concentration of fluorophores, by reducingthe frequency and thus the photon energy of the input light, or byemploying more robust fluorophores that are less prone to bleaching(e.g. Alexa Fluors or DyLight Fluors). See, e.g., Ghauharali R. et al.Journal of Microscopy 2001; 198: 88-100; and Eggeling C. et al.Analytical Chemistry 1998; 70:2651-59.

Thus, photobleaching may be used to remove, modify or in some instanceextinguish signal from a signal-emitting moiety. Photobleaching may beperformed by exposing fluorophores to a wavelength of light of suitablewavelength, energy and duration to permanently and irreversiblyextinguish the ability of the fluorophore to emit further signal.Photobleaching techniques are known in the art.

It was also found that, unlike antibodies that are generally able tobind their targets at a wide range of temperature below thephysiological temperature (i.e., 0° C. to 37° C.) and can tolerate mildvariation in salt concentration (i.e., monovalent cation concentrationfrom 10 mM to 1 M; divalent cation from 0 to 10 mM), the affinity ofshort nucleic acid hybridization is dependent on temperature and saltconcentration. For example, the predicted dissociation constant (usingthe parameter sets outlined in reference PMID 15139820) between ssDNA5′-CATCTAAAGCC-3′ and its reverse-complementary strand 5′-GGCTTTAGATG-3′is ˜90 pM at 23° C. with 500 mM [Na+] and 10 mM [Mg++] concentration. Inother words, in this condition the binding is very strong. The predicteddissociation constant of this pair of ssDNA is as high as ˜500 nM at 37°C. with 150 mM [Na+] and 0 mM [Mg++]. In other words, in this conditionthe binding is fairly weak. The dissociation constants of these twoconditions varies by nearly 4 orders of magnitude even though mostantibodies are expected to bind their target strongly in bothconditions. Similar trends are observed for other DNA sequences (FIG.10). As a further example, the imaging condition can be 23° C. with 500mM [Na+] and 10 mM [Mg++], and the dye-inactivating condition can be 37°C. with 150 mM [Na+] and 0 mM [Mg++].

In some embodiments, the sample being analyzed is cultured cells, tissuesections, or other samples from living organisms.

In some embodiments, the sample is dissociated cells that areimmobilized to a solid surface (e.g. glass slide or cover slip),including individually immobilized. For example, the sample may be cellsin blood. For example, the sample may contain cancer cells circulatingin the blood (also known as circulating tumor cells, or CTCs). Thesample may be cells grown in suspension. The sample may be cellsdisseminated from a solid tissue.

Sample

A “sample” may comprise cells (or a cell), tissue, or bodily fluid suchas blood (serum and/or plasma), urine, semen, lymphatic fluid,cerebrospinal fluid or amniotic fluid. A sample may be obtained from (orderived from) any source including, without limitation, humans, animals,bacteria, viruses, microbes and plants. In some embodiments, a sample isa cell lysate or a tissue lysate. A sample may also contain mixtures ofmaterial from one source or different sources. A sample may be a spatialarea or volume (e.g., a grid on an array, or a well in a plate or dish).A sample, in some embodiments, includes target(s), BP-NA conjugate(s)and imager strand(s). The cells may be disseminated (or dissociated)cells.

Target

A “target” is any moiety that one wishes to observe or quantitate andfor which a binding partner exists. A target, in some embodiments, maybe non-naturally occurring. The target, in some embodiments, may be abiomolecule. As used herein, a “biomolecule” is any molecule that isproduced by a living organism, including large macromolecules such asproteins, polysaccharides, lipids and nucleic acids (e.g., DNA and RNAsuch as mRNA), as well as small molecules such as primary metabolites,secondary metabolites, and natural products. Examples of biomoleculesinclude, without limitation, DNA, RNA, cDNA, or the DNA product of RNAsubjected to reverse transcription, A23187 (Calcimycin, CalciumIonophore), Abamectine, Abietic acid, Acetic acid, Acetylcholine, Actin,Actinomycin D, Adenosine, Adenosine diphosphate (ADP), Adenosinemonophosphate (AMP), Adenosine triphosphate (ATP), Adenylate cyclase,Adonitol, Adrenaline, epinephrine, Adrenocorticotropic hormone (ACTH),Aequorin, Aflatoxin, Agar, Alamethicin, Alanine, Albumins, Aldosterone,Aleurone, Alpha-amanitin, Allantoin, Allethrin, α-Amanatin, Amino acid,Amylase, Anabolic steroid, Anethole, Angiotensinogen, Anisomycin,Antidiuretic hormone (ADH), Arabinose, Arginine, Ascomycin, Ascorbicacid (vitamin C), Asparagine, Aspartic acid, Asymmetricdimethylarginine, Atrial-natriuretic peptide (ANP), Auxin, Avidin,Azadirachtin A—C35H44O16, Bacteriocin, Beauvericin, Bicuculline,Bilirubin, Biopolymer, Biotin (Vitamin H), Brefeldin A, Brassinolide,Brucine, Cadaverine, Caffeine, Calciferol (Vitamin D), Calcitonin,Calmodulin, Calmodulin, Calreticulin, Camphor—(C10H16O), Cannabinol,Capsaicin, Carbohydrase, Carbohydrate, Carnitine, Carrageenan, Casein,Caspase, Cellulase, Cellulose—(C6H10O5), Cerulenin, Cetrimonium bromide(Cetrimide)—C19H42BrN, Chelerythrine, Chromomycin A3, Chaparonin,Chitin, α-Chloralose, Chlorophyll, Cholecystokinin (CCK), Cholesterol,Choline, Chondroitin sulfate, Cinnamaldehyde, Citral, Citric acid,Citrinin, Citronellal, Citronellol, Citrulline, Cobalamin (vitamin B12),Coenzyme, Coenzyme Q, Colchicine, Collagen, Coniine, Corticosteroid,Corticosterone, Corticotropin-releasing hormone (CRH), Cortisol,Creatine, Creatine kinase, Crystallin, α-Cyclodextrin, Cyclodextringlycosyltransferase, Cyclopamine, Cyclopiazonic acid, Cysteine, Cystine,Cytidine, Cytochalasin, Cytochalasin E, Cytochrome, Cytochrome C,Cytochrome c oxidase, Cytochrome c peroxidase, Cytokine,Cytosine—C4H5N3O, Deoxycholic acid, DON (DeoxyNivalenol),Deoxyribofuranose, Deoxyribose, Deoxyribose nucleic acid (DNA), Dextran,Dextrin, DNA, Dopamine, Enzyme, Ephedrine, Epinephrine—C9H13NO3, Erucicacid—CH3(CH2)7CH═CH(CH2)11COOH, Erythritol, Erythropoietin (EPO),Estradiol, Eugenol, Fatty acid, Fibrin, Fibronectin, Folic acid (VitaminM), Follicle stimulating hormone (FSH), Formaldehyde, Formic acid,Formnoci, Fructose, Fumonisin B1, Gamma globulin, Galactose, Gammaglobulin, Gamma-aminobutyric acid, Gamma-butyrolactone,Gamma-hydroxybutyrate (GHB), Gastrin, Gelatin, Geraniol, Globulin,Glucagon, Glucosamine, Glucose—C6H12O6, Glucose oxidase, Gluten,Glutamic acid, Glutamine, Glutathione, Gluten, Glycerin (glycerol),Glycine, Glycogen, Glycolic acid, Glycoprotein, Gonadotropin-releasinghormone (GnRH), Granzyme, Green fluorescent protein, Growth hormone,Growth hormone-releasing hormone (GHRH), GTPase, Guanine, Guanosine,Guanosine triphosphate (+GTP), Haptoglobin, Hematoxylin, Heme,Hemerythrin, Hemocyanin, Hemoglobin, Hemoprotein, Heparan sulfate, Highdensity lipoprotein, HDL, Histamine, Histidine, Histone, Histonemethyltransferase, HLA antigen, Homocysteine, Hormone, human chorionicgonadotropin (hCG), Human growth hormone, Hyaluronate, Hyaluronidase,Hydrogen peroxide, 5-Hydroxymethylcytosine, Hydroxyproline,5-Hydroxytryptamine, Indigo dye, Indole, Inosine, Inositol, Insulin,Insulin-like growth factor, Integral membrane protein, Integrase,Integrin, Intein, Interferon, Inulin, Ionomycin, Ionone, Isoleucine,Iron-sulfur cluster, K252a, K252b, KT5720, KT5823, Keratin, Kinase,Lactase, Lactic acid, Lactose, Lanolin, Lauric acid, Leptin, LeptomycinB, Leucine, Lignin, Limonene, Linalool, Linoleic acid, Linolenic acid,Lipase, Lipid, Lipid anchored protein, Lipoamide, Lipoprotein, Lowdensity lipoprotein, LDL, Luteinizing hormone (LH), Lycopene, Lysine,Lysozyme, Malic acid, Maltose, Melatonin, Membrane protein,Metalloprotein, Metallothionein, Methionine, Mimosine, Mithramycin A,Mitomycin C, Monomer, Mycophenolic acid, Myoglobin, Myosin, Naturalphenols, Nucleic Acid, Ochratoxin A, Oestrogens, Oligopeptide,Oligomycin, Orcin, Orexin, Ornithine, Oxalic acid, Oxidase, Oxytocin,p53, PABA, Paclitaxel, Palmitic acid, Pantothenic acid (vitamin B5),parathyroid hormone (PTH), Paraprotein, Pardaxin, Parthenolide, Patulin,Paxilline, Penicillic acid, Penicillin, Penitrem A, Peptidase, Pepsin,Peptide, Perimycin, Peripheral membrane protein, Perosamine,Phenethylamine, Phenylalanine, Phosphagen, phosphatase, Phospholipid,Phenylalanine, Phytic acid, Plant hormones, Polypeptide, Polyphenols,Polysaccharides, Porphyrin, Prion, Progesterone, Prolactin (PRL),Proline, Propionic acid, Protamine, Protease, Protein, Proteinoid,Putrescine, Pyrethrin, Pyridoxine or pyridoxamine (Vitamin B6),Pyrrolysine, Pyruvic acid, Quinone, Radicicol, Raffinose, Renin,Retinene, Retinol (Vitamin A), Rhodopsin (visual purple), Riboflavin(vitamin B2), Ribofuranose, Ribose, Ribozyme, Ricin, RNA—Ribonucleicacid, RuBisCO, Safrole, Salicylaldehyde, Salicylic acid,Salvinorin-A—C23H28O8, Saponin, Secretin, Selenocysteine,Selenomethionine, Selenoprotein, Serine, Serine kinase, Serotonin,Skatole, Signal recognition particle, Somatostatin, Sorbic acid,Squalene, Staurosporin, Stearic acid, Sterigmatocystin, Sterol,Strychnine, Sucrose (sugar), Sugars (in general), superoxide, T2 Toxin,Tannic acid, Tannin, Tartaric acid, Taurine, Tetrodotoxin, Thaumatin,Topoisomerase, Tyrosine kinase, Taurine, Testosterone,Tetrahydrocannabinol (THC), Tetrodotoxin, Thapsigargin, Thaumatin,Thiamine (vitamin B1)—C12H17ClN4OS.HCl, Threonine, Thrombopoietin,Thymidine, Thymine, Triacsin C, Thyroid-stimulating hormone (TSH),Thyrotropin-releasing hormone (TRH), Thyroxine (T4), Tocopherol (VitaminE), Topoisomerase, Triiodothyronine (T3), Transmembrane receptor,Trichostatin A, Trophic hormone, Trypsin, Tryptophan, Tubulin,Tunicamycin, Tyrosine, Ubiquitin, Uracil, Urea, Urease, Uricacid—C5H4N4O3, Uridine, Valine, Valinomycin, Vanabins, Vasopressin,Verruculogen, Vitamins (in general), Vitamin A (retinol), Vitamin B,Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin ornicotinic acid), Vitamin B4 (adenine), Vitamin B5 (pantothenic acid),Vitamin B6 (pyridoxine or pyridoxamine), Vitamin B12 (cobalamin),Vitamin C (ascorbic acid), Vitamin D (calciferol), Vitamin E(tocopherol), Vitamin F, Vitamin H (biotin), Vitamin K (naphthoquinone),Vitamin M (folic acid), Wortmannin and Xylose.

In some embodiments, a target may be a protein target such as, forexample, proteins of a cellular environment (e.g., intracellular ormembrane proteins). Examples of proteins include, without limitation,fibrous proteins such as cytoskeletal proteins (e.g., actin, arp2/3,coronin, dystrophin, FtsZ, keratin, myosin, nebulin, spectrin, tau,titin, tropomyosin, tubulin and collagen) and extracellular matrixproteins (e.g., collagen, elastin, f-spondin, pikachurin, andfibronectin); globular proteins such as plasma proteins (e.g., serumamyloid P component and serum albumin), coagulation factors (e.g.,complement proteins, C1-inhibitor and C3-convertase, Factor VIII, FactorXIII, fibrin, Protein C, Protein S, Protein Z, Protein Z-relatedprotease inhibitor, thrombin, Von Willebrand Factor) and acute phaseproteins such as C-reactive protein; hemoproteins; cell adhesionproteins (e.g., cadherin, ependymin, integrin, Ncam and selectin);transmembrane transport proteins (e.g., CFTR, glycophorin D andscramblase) such as ion channels (e.g., ligand-gated ion channels suchnicotinic acetylcholine receptors and GABAa receptors, and voltage-gatedion channels such as potassium, calcium and sodium channels),synport/antiport proteins (e.g., glucose transporter); hormones andgrowth factors (e.g., epidermal growth factor (EGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), peptidehormones such as insulin, insulin-like growth factor and oxytocin, andsteroid hormones such as androgens, estrogens and progesterones);receptors such as transmembrane receptors (e.g., G-protein-coupledreceptor, rhodopsin) and intracellular receptors (e.g., estrogenreceptor); DNA-binding proteins (e.g., histones, protamines, CIprotein); transcription regulators (e.g., c-myc, FOXP2, FOXP3, MyoD andP53); immune system proteins (e.g., immunoglobulins, majorhistocompatibility antigens and T cell receptors); nutrientstorage/transport proteins (e.g., ferritin); chaperone proteins; andenzymes.

In some embodiments, a target may be a nucleic acid target such as, forexample, nucleic acids of a cellular environment. As used herein withrespect to targets, docking strands, and imager strands, a “nucleicacid” refers to a polymeric form of nucleotides of any length, such asdeoxyribonucleotides or ribonucleotides, or analogs thereof. Forexample, a nucleic acid may be a DNA, RNA or the DNA product of RNAsubjected to reverse transcription. Non-limiting examples of nucleicacids include coding or non-coding regions of a gene or gene fragment,loci (locus) defined from linkage analysis, exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantnucleic acids, branched nucleic acids, plasmids, vectors, isolated DNAof any sequence, isolated RNA of any sequence, nucleic acid probes, andprimers. Other examples of nucleic acids include, without limitation,cDNA, aptamers, peptide nucleic acids (“PNA”), 2′-5′ DNA (a syntheticmaterial with a shortened backbone that has a base-spacing that matchesthe A conformation of DNA; 2′-5′ DNA will not normally hybridize withDNA in the B form, but it will hybridize readily with RNA), lockednucleic acids (“LNA”), and nucleic acids with modified backbones (e.g.,base- or sugar-modified forms of naturally-occurring nucleic acids). Anucleic acid may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs (“analogous” forms of purines andpyrimidines are well known in the art). If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. A nucleic acid may be a single-stranded, double-stranded,partially single-stranded, or partially double-stranded DNA or RNA.

In some embodiments, a nucleic acid (e.g., a nucleic acid target) isnaturally-occurring. As used herein, a “naturally occurring” refers to anucleic acid that is present in organisms or viruses that exist innature in the absence of human intervention. In some embodiments, anucleic acid naturally occurs in an organism or virus. In someembodiments, a nucleic acid is genomic DNA, messenger RNA, ribosomalRNA, micro-RNA, pre-micro-RNA, pro-micro-RNA, viral DNA, viral RNA orpiwi-RNA. In some embodiments, a nucleic acid target is not a syntheticDNA nanostructure (e.g., two-dimensional (2-D) or three-dimensional(3-D) DNA nanostructure that comprises two or more nucleic acidshybridized to each other by Watson-Crick interactions to form the 2-D or3-D nanostructure).

The nucleic acid docking strands and imager strands described herein canbe any one of the nucleic acids described above (e.g., DNA, RNA,modified nucleic acids, nucleic acid analogues, naturally-occurringnucleic acids, synthetic nucleic acids).

Compositions

Provided herein are compositions that comprise at least one or at leasttwo (e.g., a plurality) BP-NA conjugate(s) (e.g., protein-nucleic acidconjugate(s)) of the invention. The BP-NA conjugates may be bound to atarget of interest (e.g., biomolecule) and/or stable bound to acomplementary fluorescently labeled imager strand. A composition maycomprise a plurality of the same species or distinct species of BP-NAconjugates. In some embodiments, a composition may comprise at least 10,50, 100, 500, 1000, 2000, 3000, 4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ BP-NA conjugates. In some embodiments, acomposition may comprise at least 10, 50, 100, 500, 1000, 2000, 3000,4000, 5000, 10⁴, 50000, 10⁵, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹complementary fluorescently labeled imager strands. In some embodiments,a composition may contain 1 to about 200 or more distinct species ofBP-NA conjugates and/or imager strands. For example, a composition maycontain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200 ormore distinct species. In some embodiments, a composition may containless than about 5 to about 200 distinct species of BP-NA conjugatesand/or imager strands. For example, a composition may contain less than5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175 or 200 distinct species.

It should be understood that the number of complementary fluorescentlylabeled imager strands imager stands in a composition may be less than,equal to or greater than the number of BP-NA conjugates in thecomposition.

Kits

The invention further provides kits comprising one or more components ofthe invention. The kits may comprise, for example, a BP-NA conjugateand/or a fluorescently labeled imager strands. The kits may alsocomprise components for producing a BP-NA conjugate or for labeling animager strand. For example, the kits may comprise a binding partner(e.g., antibody), docking strands and intermediate linkers such as, forexample, biotin and streptavidin molecules, and/or imager strands. Thekits can be used for any purpose apparent to those of skill in the art,including, those described above.

The kits may include other reagents as well, for example, buffers forperforming hybridization reactions. The kit may also includeinstructions for using the components of the kit, and/or for makingand/or using the BP-NA conjugates and/or labeled imager strands.

Applications

The BP-NA conjugates (e.g., protein-nucleic acid conjugates orantibody-nucleic acid conjugates) of the invention can be used, interalia, in any assay in which existing target detection technologies areused.

Typically assays include detection assays including diagnostic assays,prognostic assays, patient monitoring assays, screening assays,bio-warfare assays, forensic analysis assays, prenatal genomicdiagnostic assays and the like. The assay may be an in vitro assay or anin vivo assay. The present invention provides the advantage that manydifferent targets can be analyzed at one time from a single sample usingthe methods of the invention, even where such targets are spatially notresolvable (and thus spatially indistinct) using prior art imagingmethods. This allows, for example, for several diagnostic tests to beperformed on one sample.

The BP-NA conjugates can also be used to simply observe an area orregion.

The methods of the invention may be applied to the analysis of samplesobtained or derived from a patient so as to determine whether a diseasedcell type is present in the sample and/or to stage the disease. Forexample, a blood sample can be assayed according to any of the methodsdescribed herein to determine the presence and/or quantity of markers ofa cancerous cell type in the sample, thereby diagnosing or staging thecancer.

Alternatively, the methods described herein can be used to diagnosepathogen infections, for example infections by intracellular bacteriaand viruses, by determining the presence and/or quantity of markers ofbacterium or virus, respectively, in the sample. Thus, the targetsdetected using the compositions and methods of the invention may beeither patient markers (such as a cancer marker) or markers of infectionwith a foreign agent, such as bacterial or viral markers.

The quantitative imaging methods of the invention may be used, forexample, to quantify targets (e.g., target biomolecules) whose abundanceis indicative of a biological state or disease condition (e.g., bloodmarkers that are upregulated or down-regulated as a result of a diseasestate).

Further, the compositions and methods of the invention may be used toprovide prognostic information that assists in determining a course oftreatment for a patient. For example, the amount of a particular markerfor a tumor can be accurately quantified from even a small sample from apatient. For certain diseases like breast cancer, overexpression ofcertain proteins, such as Her2-neu, indicate a more aggressive course oftreatment will be needed.

The methods of the present invention may also be used for determiningthe effect of a perturbation, including chemical compounds, mutations,temperature changes, growth hormones, growth factors, disease, or achange in culture conditions, on various targets, thereby identifyingtargets whose presence, absence or levels are indicative of a particularbiological states. In some embodiments, the present invention is used toelucidate and discover components and pathways of disease states. Forexample, the comparison of quantities of targets present in a diseasetissue with “normal” tissue allows the elucidation of important targetsinvolved in the disease, thereby identifying targets for thediscovery/screening of new drug candidates that can be used to treatdisease.

The sample being analyzed may be a biological sample, such as blood,sputum, lymph, mucous, stool, urine and the like. The sample may be anenvironmental sample such as a water sample, an air sample, a foodsample and the like. The assay may be carried out with one or morecomponents of the binding reaction immobilized. Thus, the targets or theBP-NA conjugates may be immobilized. The assay may be carried out withone or more components of the binding reaction non-immobilized. Theassays may involve detection of a number of targets in a sample,essentially at the same time, in view of the multiplexing potentialoffered by the BP-NA conjugates and fluorescently labeled imager strandsof the invention. As an example, an assay may be used to detect aparticular cell type (e.g., based on a specific cell surface receptor)and a particular genetic mutation in that particular cell type. In thisway, an end user may be able to determine how many cells of a particulartype carry the mutation of interest, as an example.

Various Embodiments

This disclosure provides a variety of embodiments including but notlimited to the following numbered embodiments:

-   1. A method comprising

(1) contacting a sample being tested for the presence of one or moretargets with one or more target-specific binding partners, wherein eachtarget-specific binding partner is linked to a docking strand, andwherein target-specific binding partners of different specificity arelinked to different docking strands,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager strands having anucleotide sequence that is complementary to a docking strand,

(4) optionally removing unbound labeled imager strands,

(5) imaging the sample to detect location and number of bound labeledimager strands,

(6) extinguishing signal from the bound labeled imager strand, and

(7) repeating steps (3)-(6), each time with a labeled imager strandhaving a unique nucleotide sequence relative to all other labeled imagerstrands.

-   2. The method of embodiment 1, wherein the sample is contacted with    more than one target-specific binding partner in step (1).-   3. The method of embodiment 1 or 2, wherein the target-specific    binding partner is an antibody or an antibody fragment.-   4. The method of any one of embodiments 1-3, wherein the labeled    imager strands are labeled identically.-   5. The method of any one of embodiments 1-3, wherein the labeled    imager strands each comprise a distinct label.-   6. The method of any one of embodiments 1-5, wherein the labeled    imager strands are fluorescently labeled imager strands.-   7. The method of any one of embodiments 1-6, wherein the one or more    targets are proteins.-   8. The method of any one of embodiments 1-7, wherein the sample is a    cell, a cell lysate or a tissue lysate.-   9. The method of any one of embodiments 1-8, wherein the sample is    imaged in step (5) using confocal or epi-fluorescence microscopy.-   10. The method of any one of embodiments 1-9, wherein extinguishing    signal in step (6) comprises photobleaching.-   11. A composition comprising    -   a sample bound to more than one target-specific binding        partners, each binding partner bound to a docking strand, and    -   at least one docking strand stably bound to a labeled imager        strand.-   12. A method comprising

(1) contacting a sample being tested for the presence of one or moretargets with one or more target-specific binding partners, wherein eachtarget-specific binding partner is linked to a docking nucleic acid, andwherein target-specific binding partners of different specificity arelinked to different docking nucleic acids,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager nucleic acids having anucleotide sequence that is complementary to a docking nucleic acid,

(4) optionally removing unbound labeled imager nucleic acids,

(5) imaging the sample to detect location and number of bound labeledimager nucleic acids,

(6) removing the bound labeled imager nucleic acids from the dockingnucleic acids, and

(7) repeating steps (3)-(6), each time with a labeled imager nucleicacid having a unique nucleotide sequence relative to all other labeledimager nucleic acids.

-   13. The method of embodiment 12, wherein the sample is contacted    with more than one target-specific binding partner in step (1).-   14. The method of embodiment 12 or 13, wherein the target-specific    binding partner is an antibody or an antibody fragment.-   15. The method of embodiment 12 or 13, wherein the target-specific    binding partner is a natural or engineered ligand, a small molecule,    an aptamer, a peptide or an oligonucleotide.-   16. The method of any one of embodiments 12-15, wherein the labeled    imager nucleic acids are labeled identically.-   17. The method of any one of embodiments 12-15, wherein the labeled    imager nucleic acids each comprise a distinct label.-   18. The method of any one of embodiments 12-17, wherein the labeled    imager nucleic acids are fluorescently labeled imager nucleic acids.-   19. The method of any one of embodiments 12-18, wherein the one or    more targets are proteins.-   20. The method of any one of embodiments 12-19, wherein the sample    is a cell, a cell lysate or a tissue lysate.-   21. The method of any one of embodiments 12-20, wherein the sample    is imaged in step (5) using confocal or epi-fluorescence microscopy.-   22. The method of any one of embodiments 12-21, wherein the labeled    imager nucleic acids are removed from the docking nucleic acids by    decreasing salt concentration, addition of a denaturant, or    increasing temperature.-   23. The method of embodiment 22, wherein the salt is Mg++.-   24. The method of embodiment 22, wherein the denaturant is    formamide, urea or DMSO.-   25. The method of any one of embodiments 12-21, wherein the labeled    imager nucleic acids are not removed from the docking nucleic acids    by strand displacement in the presence of a competing nucleic acid.-   26. The method of any one of embodiments 12-21, wherein the labeled    imager nucleic acids are removed from the docking nucleic acids by    chemically, photochemically, or enzymatically cleaving, modifying or    degrading the labeled imager nucleic acids.-   27. The method of any one of embodiments 12-21, wherein, when the    labeled imager nucleic acid is bound to its respective docking    nucleic acid, there is no single-stranded region on the imager    nucleic acid or the docking nucleic acid.-   28. The method of any one of embodiments 12-21, wherein the labeled    imager nucleic acid is not self-quenching.-   29. The method of any one of embodiments 12-28, wherein the unbound    docking nucleic acid is partially double-stranded.-   30. The method of any one of embodiments 12-28, wherein the unbound    imager nucleic acid is partially double-stranded.-   31. The method of any one of embodiments 12-28, wherein the imager    nucleic acid is a molecular beacon or comprises a hairpin secondary    structure.-   32. The method of any one of embodiments 12-27 and 29-31, wherein    the imager nucleic acid is a molecular beacon or comprises a hairpin    secondary structure that is self-quenching.-   33. The method of any one of embodiments 12-28, wherein the imager    nucleic acid is a hemiduplex.-   34. The method of embodiment 33, wherein the hemiduplex is    self-quenching.-   35. The method of any one of embodiments 12-34, wherein the docking    nucleic acid comprises a hairpin secondary structure.-   36. The method of any one of embodiments 12-35, wherein the imager    nucleic acid is bound to multiple signal-emitting moieties through a    dendrimeric structure or a polymeric structure.-   34. A method comprising

(1) contacting a sample being tested for the presence of one or moretargets with one or more target-specific binding partners, wherein eachtarget-specific binding partner is linked to a docking nucleic acid, andwherein target-specific binding partners of different specificity arelinked to different docking nucleic acids,

(2) optionally removing unbound target-specific binding partners,

(3) contacting the sample with labeled imager nucleic acids having anucleotide sequence that is complementary to a docking nucleic acid,

(4) optionally removing unbound labeled imager nucleic acids,

(5) imaging the sample to detect location and number of bound labeledimager nucleic acids,

(6) inactivating the bound labeled imager nucleic acids, by removing ormodifying their signal-emitting moieties without removing the imagernucleic acid in its entirety, and

(7) repeating steps (3)-(6), each time with a labeled imager nucleicacids having a unique nucleotide sequence relative to all other labeledimager nucleic acids.

-   35. The method of embodiment 34, wherein the sample is contacted    with more than one target-specific binding partner in step (1).-   36. The method of embodiment 34 or 35, wherein the target-specific    binding partner is an antibody or an antibody fragment.-   37. The method of embodiment 34 or 35, wherein the target-specific    binding partner is a natural or engineered ligand, a small molecule,    an aptamer, a peptide or an oligonucleotide.-   38. The method of any one of embodiments 34-37, wherein the labeled    imager nucleic acids are labeled identically.-   39. The method of any one of embodiments 34-37, wherein the labeled    imager nucleic acids each comprise a distinct label.-   40. The method of any one of embodiments 34-39, wherein the labeled    imager nucleic acids are fluorescently labeled imager nucleic acids.-   41. The method of any one of embodiments 34-40, wherein the one or    more targets are proteins.-   42. The method of any one of claims 34-41, wherein the sample is a    cell, a cell lysate or a tissue lysate.-   43. The method of any one of embodiments 34-42, wherein the sample    is imaged in step (5) using confocal or epi-fluorescence microscopy.-   44. The method of any one of embodiments 34-43, wherein the unbound    docking nucleic acid is partially double-stranded.-   45. The method of any one of embodiments 34-43, wherein the unbound    imager nucleic acid is partially double-stranded.-   46. The method of any one of embodiments 34-45, wherein the imager    nucleic acid is a molecular beacon or comprises a hairpin secondary    structure.-   47. The method of any one of embodiments 34-45, wherein the imager    nucleic acid is a molecular beacon or comprises a hairpin secondary    structure that is self-quenching.-   48. The method of any one of embodiments 34-45, wherein the imager    nucleic acid is a hemiduplex.-   49. The method of embodiment 48, wherein the hemiduplex is    self-quenching.-   50. The method of any one of embodiments 34-49, wherein the docking    nucleic acid comprises a hairpin secondary structure.-   51. The method of any one of embodiments 34-50, wherein the imager    nucleic acid is bound to multiple signal-emitting moieties through a    dendrimeric structure or a polymeric structure.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method comprising (1) contacting a sample beingtested for the presence of one or more targets with one or moretarget-specific binding partners, wherein each target-specific bindingpartner is linked to a docking strand, and wherein target-specificbinding partners of different specificity are linked to differentdocking strands to produce one or more targets bound to one or moretarget-specific binding partners, (2) optionally removing unboundtarget-specific binding partners, (3) contacting the sample with labeledimager strands that bind to docking strands, (4) optionally removingunbound labeled imager strands, (5) imaging the sample to detect boundlabeled imager strands, (6) inactivating the bound labeled imagerstrands, by removing or modifying their signal-emitting moieties withoutremoving the imager strand in its entirety, and (7) repeating at leastsome of steps (3)-(6) at least once with a labeled imager strand havinga unique composition relative to at least one other labeled imagerstrand of step (3).
 2. The method of claim 1, wherein the sample iscontacted with more than one target-specific binding partner in step(1).
 3. The method of claim 1, wherein the target-specific bindingpartner is an antibody or an antibody fragment.
 4. The method of claim1, wherein the target-specific binding partner is a ligand, a smallmolecule, an aptamer, a peptide or an oligonucleotide.
 5. The method ofclaim 1, wherein the labeled imager strands are labeled identically. 6.The method of claim 1, wherein the labeled imager strands each comprisea distinct label.
 7. The method of claim 1, wherein the labeled imagerstrands are fluorescently labeled imager strands.
 8. The method of claim1, wherein the one or more targets are proteins.
 9. The method of claim1, wherein the sample is a cell, a cell lysate or a tissue lysate. 10.The method of claim 1, wherein the sample is imaged in step (5) usingconfocal or epi-fluorescence microscopy.
 11. The method of claim 1,wherein the unbound docking strand is partially double-stranded.
 12. Themethod of claim 1, wherein the unbound imager strand is partiallydouble-stranded.
 13. The method of claim 1, wherein the imager strand isa molecular beacon or comprises a hairpin secondary structure.
 14. Themethod of claim 1, wherein the imager strand is a molecular beacon orcomprises a hairpin secondary structure that is self-quenching.
 15. Themethod of claim 1, wherein the imager strand is a hemiduplex.
 16. Themethod of claim 15, wherein the hemiduplex is self-quenching.
 17. Themethod of claim 1, wherein the docking strand comprises a hairpinsecondary structure.
 18. The method of claim 1, wherein the imagerstrand is bound to multiple signal-emitting moieties through adendrimeric structure or a polymeric structure.
 19. The method of claim1, wherein the removal or modification of the signal-emitting moietiesin step (6) comprises cleaving a linker between the imager strand andthe signal-emitting moiety.
 20. The method of claim 1, wherein theremoval or modification of the signal-emitting moieties in step (6)comprises chemically or photochemically modifying the signal-emittingmoiety.
 21. The method of claim 1, wherein the removal or modificationof the signal-emitting moieties in step (6) comprises bleaching bychemical agents.
 22. The method of claim 1, wherein the removal ormodification of the signal-emitting moieties in step (6) comprisesphotobleaching.